In an animal cell culture, when do we consider a cell to have passed from passage Pn to passage Pn+1? Consider a scenario where I've a flask containing P20 (twentieth passage) of a cell line. I trypsinise the cells, pellet them and distribute them into three more flasks. The cells in the three new flasks will be P21 (twenty-first passage) cells. Suppose some cells fail to detach during trypsinisation and are provided with media so that they grow to confluence once again. What is the passage status of these cells that were left behind due to partial trypsinisation? Are these P20 cells or P21 cells?
Simply, passage numbers are counted by number of subculture. Therefore, Regrown-remainders are also P21 cell, because it was also stimulated by trypsin and proliferated as a new batch. Trypsin-induced proteome alteration could be happend during cell subculture in mammalian cells.
Simply, passage numbers are counted by number of subculture. Therefore, Regrown-remainders are also P21 cell, because it was also stimulated by trypsin and proliferated as a new batch. Trypsin-induced proteome alteration could be happend during cell subculture in mammalian cells.
Also important to remember: passage number is quite an arbitrary metric for cell behaviour. It is...sort of used as a measure of cell age/replicative cycles, on the assumption that cell behaviour changes the further cells get from their progenitor population, but it really isn't this at all. It's a pretty bad metric, really, but we use it primarily because it is a metric we can actually count.
For example, if I split cells lightly (say, 1:2 dilution) I can rocket through the passage numbers with a concomitant increase in cell divisions. If I split cells hard (say, 1:10) then I will be racking up the cell divisions while my passage number remains modest.
Some cells, of course, are very fussy about how many friends they have nearby (die if less than 40% confluent, die if more than 80% confluent, etc: we've all had cells like this), so the extent of replication will vary from cell line to cell line, and (often) from experiment to experiment.
There's not a lot you can do about this, of course, but it's simply something to be aware of.
The cells left behind also would be P21. They no longer are P20, as the original population was reduced by the splitting of the P20 culture, so those remnants in the original plate would be identified as P21.
As John Hilyard already pointed out, passage number is a very loose and arbitrary scale for monitoring the "age" of the culture and has little correlation with total population doublings. If cell counts are done during every passage, then a rough estimation of population doublings can be tracked over time.
I agree with Hrstka, even if the cells did not detach they should be considered a new passage: these cells were not only separated from the initial population, but were also exposed to trypsin, even if they did not detach.
If you are worried about when you need to get a fresh batch of cells, I'd say the most important thing is to pay attention to the characteristics specific to your cell line and how it changes over time, such as cell morphology and behavior (how fast they proliferate, how adherent they are etc). As Hildyard mentioned, different cell lines will have distinct "expiration dates". So whenever you see a noticeable change, it's time to get new cells.
Thank you Drs. Young-Doug Sohn , John Hildyard , Ron Hrstka and Rafaela Marocci .
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